David G. Walters

The role of metabolism in 2-methoxyethanol-induced testicular toxicity.

The role of metabolism in 2-methoxyethanol (ME)-induced testicular toxicity has been investigated with Sprague-Dawley rats. Following administration of [14C]ME (250 mg/kg, ip) to a group of animals, there was evidence of testicular damage, identified as depletion of the spermatocyte population. Radioactivity detected in urine over 48 hr after treatment accounted for 55% of the dose. The major urinary metabolites were identified by HPLC and isotope dilution analysis, as methoxyacetic acid (MAA) and methoxyacetylglycine (accounting for 50 to 60% and 18 to 25%, respectively, of urinary radioactivity). Analysis of plasma revealed a rapid conversion of ME to MAA (t1/2 for disappearance of ME = 0.6 +/- 0.03 hr) and gradual clearance of radioactivity (t1/2 = 19.7 +/- 2.3 hr). Pretreatment of animals with pyrazole (400 mg/kg, ip) 1 hr prior to [14C]ME dosing gave complete protection against the testicular toxicity of ME. Radioactivity detected in the urine from the pyrazole-pretreated groups over 48 hr (18%) was significantly lower than in the ME-only group. The major radioactive peak co-chromatographed with ME (30 to 36% of the total urinary radioactivity). MAA and methoxyacetylglycine were not major metabolites. Analysis of plasma revealed almost complete inhibition of the conversion of ME to MAA (t1/2 for disappearance of ME = 42.6 +/- 5.6 hr, clearance of radioactivity t1/2 = 51.0 +/- 7.8 hr). The results demonstrate that metabolic activation is required for 2-methoxyethanol to exert toxicity to the male reproductive system.

Metabolism of coumarin and 7-ethoxycoumarin by rat, mouse, guinea pig, Cynomolgus monkey and human precision-cut liver slices

1. The metabolism of 50 microM 7-ethoxycoumarin and 50 microM [3-14C]coumarin has been studied in precision-cut liver slices from the male Sprague-Dawley rat, female DBA/2 mouse, male Dunkin-Hartley guinea pig, male Cynomolgus monkey and man. 2. In liver slices from all five species 7-ethoxycoumarin was metabolized to 7-hydroxycoumarin (7-HC), which was extensively conjugated with D-glucuronic acid and sulphate. In rat and mouse, 7-HC was preferentially conjugated with sulphate, whereas rates of glucuronidation and sulphation were similar in the other three species. 3. [3-14C]coumarin was metabolized by liver slices from all five species to various polar products and to metabolite(s) that bound covalently to liver slice proteins. In Cynomolgus monkey and both human subjects studied, 7-HC was the major metabolite that was conjugated with D-glucuronic acid and sulphate, whereas in rat the major metabolites were products of the 3-hydroxylation pathway and unknown metabolites. Major metabolites in mouse liver slices were 7-HC, 3-hydroxylation pathway products and unknown metabolites, and in guinea pig liver slices, 7-HC and unknown metabolites. 4. The metabolism of 7-ethoxycoumarin to free and conjugated 7-HC and [3-14C]coumarin to total polar products was greater in liver slices from mouse and Cynomolgus monkey than the other three species. 5. With liver slices from all five species there appeared to be little difference in the extent of metabolism of 7-ethoxycoumarin and [3-14C]coumarin to various products in either a complex tissue culture medium (RPMI 1640 plus foetal calf serum) or a simple balanced salt solution (Earles balanced salt solution). 6. These results demonstrate that precision-cut liver slices are a valuable in vitro model system for investigating species differences in xenobiotic metabolism. Generally, the observed species differences in coumarin metabolism in vitro agree well with available in vivo data.

Studies on the metabolism of deoxynivalenol in the rat

The metabolism and tissue distribution of [14C]deoxynivalenol have been studied in male PVG rats. Following administration of a single oral 10-mg/kg dose, radioactivity excreted in the urine and faeces accounted, respectively, for 25 and 64% of the administered dose within 96 hr. Less than 0.15% of the dose was detected in the respired air. Very little radioactivity appeared to be retained in any of the tissues examined after 96 hr. HPLC separation of several urinary and faecal metabolites was achieved on a reversed-phase column, using two different elution systems, one at neutral pH and one acidified. Two of the major non-polar HPLC peaks were identified by gas chromatography-mass spectrometry as unchanged deoxynivalenol and 3 alpha,7 alpha,15-trihydroxytrichothec-9,12-dien-8-one.

Metabolism of [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, syrian hamster, gerbil and humans

The metabolism of 0.19 and 2.0 mM-[3-14C]coumarin to polar products and covalently bound metabolites has been studied with hepatic microsomes from the rat, Syrian hamster, Mongolian gerbil and humans. [3-14C]Coumarin was metabolized by liver microsomes from all species to a number of polar products and to metabolite(s) that became covalently bound to microsomal proteins. The polar products included 3-, 5- and 7-hydroxycoumarins, o-hydroxyphenylacetaldehyde and o-hydroxyphenylacetic acid. Coumarin 7-hydroxylation was observed in all species except the rat. With 0.19 mM-[3-14C]coumarin, 7-hydroxycoumarin was the major metabolite in human liver microsomes, whereas in the other species with 0.19 mM substrate and in all species with 2.0 mM substrate o-hydroxyphenylacetaldehyde was the major metabolite. Of the three animal species studied the gerbil most resembled humans as this species also had a high coumarin 7-hydroxylase activity. The administration of Aroclor 1254 to the rat and Syrian hamster induced both microsomal cytochrome P-450 content and [3-14C]coumarin metabolism. With liver microsomes from all species a good correlation between rates of [3-14C]coumarin metabolism and covalent binding was observed at both substrate concentrations. However, in view of the known species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity, the present data are not consistent with microsomal coumarin metabolite covalent binding being an indicator of potential liver damage.

Effect of DI-n-pentyl phthalate treatment on testicular steroidogenic enzymes and cytochrome P-450 in the rat

Treatment of young male rats with dipentyl phthalate (DPP) produced significant decreases in testicular cytochrome P-450, cytochrome P-450 dependent microsomal steroidogenic enzymes (17 alpha-hydroxylase, 17-20 lyase) and in the maximal binding of a natural substrate (progesterone) to testis microsomes. No effect was demonstrated by this compound on hepatic cytochrome P-450 content. Treatment of animals with a phthalate ester not causing testicular atrophy (diethyl phthalate; DEP) produced no significant changes in any of the parameters measured. This effect on the enzymes responsible for androgen production may be important as a mechanism of action involved in the development of phthalate-induced testicular damage.

Identification of o-hydroxyphenylacetaldehyde as a major metabolite of coumarin in rat hepatic microsomes

The metabolism of [3-14C]coumarin has been studied in hepatic microsomes from control (corn-oil treated) and Aroclor 1254-treated (100 mg/kg body weight/day, 5 days, ip) rats. [3-14C]Coumarin metabolites in incubate extracts were separated by HPLC and identified by comparison with the retention times of known coumarin metabolites. The major product produced by incubation of 0.25-2.5 mM-[3-14C]coumarin with both control and Aroclor 1254-induced hepatic microsomes was a novel coumarin metabolite. This novel metabolite was extracted from pooled microsomal incubations, purified by semi-preparative HPLC and identified by mass spectrometry as o-hydroxyphenylacetaldehyde (o-HPA). Some possible pathways for the formation of o-HPA from coumarin are proposed.

Comparison of the metabolism of 7-ethoxycoumarin and coumarin in precision-cut rat liver and lung slices

The metabolism of 7-ethoxycoumarin and [3-(14)C]coumarin was compared in precision-cut rat liver and lung slices. The lung slices were prepared using an agarose gel instilling technique enabling the production of tissue cylinders followed by lung slices employing a Krumdieck tissue slicer. Both 50 microM 7-ethoxycoumarin and 50 microM [3-(14)C]coumarin were metabolized by rat liver and lung slices. 7-Ethoxycoumarin was converted to 7-hydroxycoumarin (7-HC) which was conjugated with both D-glucuronic acid and sulfate. 7-HC sulfate was the major metabolite formed by both liver and lung slices. [3-(14)C]Coumarin was metabolized by rat liver and lung slices to both polar products and to metabolite(s) that bound covalently to tissue slice proteins. The polar products included unidentified metabolites and 3-hydroxylation pathway products, with only very small quantities of 7-HC being formed. These results demonstrate that precision-cut lung slices are a useful model in vitro system for studying the pulmonary metabolism of xenobiotics. Moreover, the precision-cut tissue slice technique may be employed for comparisons of hepatic and extrahepatic xenobiotic metabolism.

Comparison of the metabolism and disposition of [3-14C]coumarin in the rat and marmoset (Callithrix jacchus)

Male Sprague-Dawley rats and marmosets were given a single oral 25 mg/kg dose of [3-14C]coumarin and the excretion of radioactivity in the expired air, urine and faeces monitored up to 96 h. Excretion profiles were similar in both species with the bulk of the dose being excreted in the urine and faeces within 24 h. Chromatographic analysis of 0-48 h urine samples revealed similar metabolic profiles with only small amounts of unchanged coumarin and very little 7-hydroxycoumarin. Coumarin 7-hydroxylase activity was not detectable in hepatic microsomes from either species. These results demonstrate that the disposition of [3-14C]coumarin was similar in the rat and marmoset, a New World primate, and that both species, unlike man, are poor 7-hydroxylators of coumarin.

Studies on the disposition, metabolism and hepatotoxicity of coumarin in the rat and Syrian hamster

The hepatotoxicity, metabolism and disposition of coumarin has been compared in male Sprague-Dawley rats and Syrian hamsters. The treatment of rats for 12, 24 and 42 weeks with diets containing 0.2 and 0.5% coumarin resulted in hepatotoxicity and increased relative liver weights. While levels of cytochrome P450 (CYP) and CYP-dependent enzymes were decreased, levels of reduced glutathione (GSH) and activities of UDP glucuronosyltransferase, gamma-glutamyltransferase and GSH S-transferase were increased. In contrast, coumarin produced few hepatic changes in the Syrian hamster. Following a single oral dose of 25 mg/kg [3-14C]coumarin, radioactivity was rapidly excreted by the rat and Syrian hamster with the urine containing 63.5 and 89.9%, respectively, and the faeces 38.0 and 12.4%, respectively, of the administered dose after 96 h. The biliary excretion of radioactivity was greater in the rat than in the Syrian hamster. Analysis of 0-24-h urine samples revealed that both species were poor 7-hydroxylators of coumarin. In the rat, treatment with 0.5% coumarin in the diet for 24 weeks was found to increase the urinary excretion of single oral gavage doses of 25 and 300 mg/kg [3-14C]coumarin. The marked species difference in hepatotoxicity between the rat and Syrian hamster observed in this study may be at least partially attributable to differences in coumarin disposition. However, additional studies are required to elucidate the metabolic pathways of coumarin in both species.

Metabolism of coumarin by precision-cut calf liver slices and calf liver microsomes

1. The metabolism of 50 microM [3-14C]coumarin has been studied in precision-cut-calf liver slices. 2. The metabolism of 50 microM coumarin to 7-hydroxycoumarin has also been examined in calf, rat, Cynomolgus monkey and human liver microsomal preparations. 3. In precision-cut calf liver slices, [3-14C]coumarin was metabolized to various polar products and to metabolite(s) that bound covalently to calf liver slice proteins. The polar products included 7-hydroxycoumarin (which was extensively conjugated with D-glucuronic acid and/or sulphate), metabolites of the 3-hydroxylation pathway (mainly o-hydroxyphenylethanol and o-hydroxyphenylacetic acid), and unknown metabolites. 4. Coumarin 7-hydroxylase activity was readily detectable in calf, Cynomolgus monkey and human liver microsomes, but only barely detectable in rat liver microsomes. Enzyme activity in calf, Cynomolgus monkey and human liver microsomes was inhibited by 8-methoxypsoralen (methoxsalen) with IC50s (concentration required to produce a 50% inhibition of enzyme activity) ranging from 0.3 to 2.8 microM. 5. These results and those of other studies demonstrate that precision-cut liver slices are a valuable in vitro model system for investigating species differences in xenobiotic metabolism. Coumarin is metabolized in calf liver by various pathways including both 3- and 7-hydroxylation. The inhibition of coumarin 7-hydroxylase activity by 8-methoxypsoralen suggests that calf liver microsomes contain P450A isoenzyme(s) similar to mouse 2A5 and human 2A6.